Several transcriptional factors have been implicated in human embryonic stem cell (hESC) self-renewal supporting a view that this process is regulated at the level of transcriptional control (Chambers, Cloning Stem Cells 6:386-391, 2004).
The transcription factor POU5F1 (OCT4) is essential for embryonic stem cell (ESC) pluripotency and appears to regulate a number of ESC properties. OCT4 is specifically expressed in ESCs, pre-implantation embryos, epiblast, and germ cells (Okamoto et al., Cell 60:461-472, 1990; Scholer et al., EMBO J. 9:2185-2195, 1990). Inactivation of OCT4 in mouse embryos and ESCs results in loss of pluripotency and spontaneous differentiation into the trophoblast lineage (Niwa et al., Nat. Genet. 24:372-376, 2000). Mouse ESCs (mESCs), even when constitutively expressing OCT4 from an exogenous promoter, still require LIF for self-renewal suggesting that LIF and OCT4 function in different pathways. However, overexpression of OCT4 induces mESCs to differentiate into PE (Niwa et al., Nat. Genet. 24:372-376, 2000).
The homeodomain-containing transcription factor NANOG is another critical ESC factor recently identified (Chambers et al., Cell 113:643-655, 2003; Mitsui et al., Cell 113:631-642, 2003). The NANOG-deficient ICM fails to generate an epiblast and only produces extraembryonic primitive endoderm (PE). Similarly in culture, NANOG-deficient ESCs lose pluripotency and differentiate to a PE lineage. Unlike POU5F1/OCT4, NANOG overexpression can maintain ESC self-renewal without LIF (Chambers et al., Cell 113:643-655, 2003). It has been proposed that NANOG maintains ESC self-renewal through repression of genes that promote differentiation, e.g., GATA4 and GATA6, which are upregulated in NANOG-deficient cells. That NANOG also binds sequences in the GATA6 gene supports this hypothesis (Mitsui et al., Cell 113:631-642, 2003).
Together, these observations suggest that NANOG is a critical factor underlying pluripotency in both ICM and ESCs by repressing their differentiation into PE, and that NANOG and OCT4 work together in the maintenance of the undifferentiated state by virtue of overlapping functions. Two cell fate decisions have to be made during pre-implantation development. The first is that cells of the morula remain pluripotent or differentiate into trophectoderm. The second is that cells of the ICM remain pluripotent as epiblast or differentiate into PE. OCT4 is the key determinant of the first decision (since OCT4-null ESCs differentiate into trophectoderm), while NANOG is the crucial determinant of the second decision (since ESCs lacking NANOG differentiate into PE) (Mitsui et al., Cell 113:631-642, 2003). FIG. 1 shows transcription factors involved in controlling self-renewal of human embryonic stem cells by repressing early lineage commitment.
Two other transcription factors have been identified that interact with OCT4: the forkhead transcription factor FOXD3 and the Sry-related factor SOX2. FOXD3 is expressed in the blastocyst and later in the post-implantation egg cylinder epiblast. FOXD3 physically interacts with OCT4 to activate the ostopontin enhancer, which is expressed in ESCs (Guo et al., Proc. Natl. Acad. Sci. U.S.A. 99:3663-3667, 2002). Sox2 is expressed in ESCs as well as in multipotent embryonic and extra-embryonic lineages. Disrupting Sox2 results in pre-implantation embryonic lethality (Avilion et al., Genes Dev. 17:126-140, 2003). Sox2 was identified as a co-factor of OCT4 for activating FGF4, which is restrictively expressed in undifferentiated ESCs, and is essential for post-implantation mouse development and limb patterning and growth (Yuan et al., Genes Dev. 9:2635-2645, 1995). Transcriptional regulation of NANOG itself is also regulated by OCT4 and SOX2 (Rodda et al., J. Biol. Chem. 280: 24731-24737, 2005). Another OCT4 and SOX2 co-regulated gene is the ESC-specific transcription factor UTF1 (Nishimoto et al., Mol. Cell. Biol. 19:5453-5465, 1999). Taken together, these studies suggest that the SOX2-OCT4 complex is at the apex of a regulatory hierarchy of the “pluripotency genetic regulatory network”.
FIG. 1 shows transcription factors involved in controlling self-renewal by repressing early lineage commitment.
In summary, ESC identity is determined by cell-intrinsic transcription factors that need to be expressed at particular levels in order to function appropriately. However, the molecular basis of the regulation of pluripotency and early lineage commitment of hESCs is still poorly understood. Additional intrinsic pathway-specific transcription factors presumably exist that maintain expression of the thousands of genes that are expressed in ESCs and control different types of renewal and differentiation pathways. Understanding how hESCs maintain their pluripotency and self-renewal and execute precise differentiation programs will require extending our understanding of the transcriptional regulatory hierarchy of hESC function, including identifying new intrinsic transcription factors.